Activation of the extracellular signal-regulated kinases 1 and 2 by glial cell line-derived neurotrophic factor and its relation to neuroprotection in a mouse model of Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to be neuroprotective in animal models of the dopamine deficiency in Parkinson's disease. To examine the role of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in this process, we infused a single dose of GDNF into the striatum of mice and analyzed the effect on ERK1/2 by immunohistochemistry and Western blot analysis. GDNF caused an increase in the phosphorylation of ERK1/2 both in the striatum and in tyrosine hydroxylase-positive neurons in the substantia nigra. In the striatum, the increase in ERK1/2 phosphorylation was evident by 3 hr and persisted for at least 7 days, whereas, in the substantia nigra, an increase in phosphorylated ERK1/2 was first evident at 24 hr and persisted for at least 7 days. The increase in phosphorylated ERK1/2 was maximal at 0.45 microg GDNF at the time points examined. GDNF also protected dopamine terminals against the loss of tyrosine hydroxylase immunoreactivity normally associated with the intrastriatal administration of 6-hydroxydopamine (0.5 microg/0.5 microl). However, this was observed only at a much higher dose of GDNF, 4.5 microg. Thus, our results suggest that the ability of GDNF to protect dopamine neurons cannot be explained solely in terms of its influence on ERK1/2 and that the role of other signaling pathways should be explored.

A model of GDNF gene therapy in mice with 6-Hydroxydopamine lesions: time course of Neurorestorative effects and ERK1/2 activation.

BACKGROUND: Glial cell line-derived neurotrophic factor (GDNF) is the most promising neurotrophin for restorative treatments in Parkinson's disease, but its biological effects are not completely understood. OBJECTIVE: To define a model of GDNF gene therapy in the mouse, we studied the long-term effects of lentiviral GDNF delivery in mice with striatal 6-hydroxydopamine (6-OHDA) lesions. METHODS: Lentiviral vectors coding for GDNF or green fluorescent protein (GFP) were injected unilaterally in the striatum two weeks prior to the 6-OHDA lesion. Mice were monitored on tests of spontaneous activity and amphetamine-induced rotation at 1, 4, 10 and 35 weeks post-lesion. Brains were processed immunohistochemically for tyrosine hydroxylase (TH) and markers of extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation at the same time points. RESULTS: Lentiviral GDNF significantly inhibited both spontaneous and amphetamine-induced rotation. Compared to the control vector, lentiviral GDNF resulted in a partial protection of TH-positive cells in the substantia nigra, and in a nearly total restoration of striatal TH immunostaining by 35 weeks. A progressive sprouting of TH-positive neurites occurred in both the globus pallidus and the substantia nigra, reaching a 4-5 fold increase above controls by 35 weeks. This effect was paralleled by a long-term supranormal activation of ERK1/2 and its downstream target, phospho-Ser31 TH. CONCLUSIONS: Lentiviral GDNF delivery produced robust long-term signaling responses and neurorestoration. This experimental model of GDNF gene therapy will be particularly suitable to study the molecular mechanisms of dopaminergic fiber sprouting, a long-term response to GDNF delivery that also occurs in Parkinson's disease patients.

Activation of extracellular signal-regulated kinases 1 and 2 by depolarization stimulates tyrosine hydroxylase phosphorylation and dopamine synthesis in rat brain.

Production of dopamine is regulated via phosphorylation of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines. Here we have used a preparation of rat striatal slices to examine the involvement of two mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), in the depolarization-dependent regulation of TH phosphorylation and dopamine synthesis. Depolarization with elevated KCl (45 mm) caused an increase in the phosphorylation state and, thereby, activation of ERK1/2. The same stimulus also increased TH phosphorylation at Ser19, Ser31 and Ser40 (measured using site- and phospho-specific antibodies) and TH activity [measured as 3,4-dihydroxyphenylalanine (DOPA) accumulation]. A MAPK/ERK kinase inhibitor, PD098059, decreased the basal levels of phospho-ERK1/2 and prevented the increase in ERK1/2 phosphorylation induced by depolarization. PD098059 also decreased both basal and depolarization-induced phosphorylation of TH at Ser31 and reduced the increase in Ser40 phosphorylation induced by high potassium, but did not affect Ser19 phosphorylation. PD098059 alone inhibited basal TH activity and decreased the accumulation of DOPA induced by depolarization. These data provide evidence for the involvement of ERK1/2 in the regulation of the state of phosphorylation of TH at Ser31 and Ser40 and a correlation between ERK1/2-dependent phosphorylation of TH and stimulation of dopamine synthesis in the brain.

Distinct roles of dopamine D2L and D2S receptor isoforms in the regulation of protein phosphorylation at presynaptic and postsynaptic sites.

Dopamine D2 receptors are highly expressed in the dorsal striatum where they participate in the regulation of (i) tyrosine hydroxylase (TH), in nigrostriatal nerve terminals, and (ii) the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), in medium spiny neurons. Two isoforms of the D2 receptor are generated by differential splicing of the same gene and are referred to as short (D2S) and long (D2L) dopamine receptors. Here we have used wild-type mice, dopamine D2 receptor knockout mice (D2 KO mice; lacking both D2S and D2L receptors) and D2L receptor-selective knockout mice (D2L KO mice) to evaluate the involvement of each isoform in the regulation of the phosphorylation of TH and DARPP-32. Incubation of striatal slices from wild-type mice with quinpirole, a dopamine D2 receptor agonist, decreased the state of phosphorylation of TH at Ser-40 and its enzymatic activity. Both effects were abolished in D2 KO mice but were still present in D2L KO mice. In wild-type mice, quinpirole inhibits the increase in DARPP-32 phosphorylation at Thr-34 induced by SKF81297, a dopamine D1 receptor agonist. This effect is absent in D2 KO as well as D2L KO mice. The inability of quinpirole to regulate DARPP-32 phosphorylation in D2L KO mice cannot be attributed to decreased coupling of D2S receptors to G proteins, because quinpirole produces a similar stimulation of [(35)S]GTPgammaS binding in wild-type and D2L KO mice. These results demonstrate that D2S and D2L receptors participate in presynaptic and postsynaptic dopaminergic transmission, respectively.